The disclosure provides methods of synthesizing DNA using topoisomerase-mediated ligation, by adding single nucleotides or oligomers to a DNA strand in the 3′ to 5′ direction.
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1. A method of synthesizing a DNA molecule using topoisomerase-mediated ligation, by adding single nucleotides or oligomers to a DNA strand in the 3′ to 5′ direction, comprising (i) reacting a DNA molecule with a topoisomerase charged with the desired nucleotide or oligomer wherein the nucleotide or oligomer is blocked from further addition at the 5′ end, then (ii) deblocking the 5′ end of the DNA thus formed, and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained.
This invention relates to a method for synthesizing DNA molecules using topoisomerase-mediated ligation, addressing the need for efficient and precise DNA assembly. The process involves adding single nucleotides or oligomers to a DNA strand in the 3′ to 5′ direction through a controlled enzymatic reaction. The method begins by reacting a DNA molecule with a topoisomerase that is pre-loaded (charged) with the desired nucleotide or oligomer. The nucleotide or oligomer is initially blocked at the 5′ end to prevent further uncontrolled additions. After the ligation step, the 5′ end is deblocked, allowing the next nucleotide or oligomer to be added. These steps are repeated iteratively until the desired DNA sequence is fully synthesized. The use of topoisomerase ensures precise and efficient ligation, while the blocking and deblocking steps provide control over the synthesis process. This approach enables the assembly of custom DNA sequences with high fidelity and minimal errors, making it suitable for applications in synthetic biology, genetic engineering, and molecular cloning.
2. The method of claim 1 wherein single nucleotides are added.
This invention relates to a method for sequencing nucleic acids, specifically DNA or RNA, by synthesizing complementary strands while detecting the incorporation of individual nucleotides. The method addresses the challenge of accurately sequencing long nucleic acid molecules by ensuring precise detection of each nucleotide addition during synthesis. The process involves providing a nucleic acid template, a primer, and a polymerase enzyme capable of synthesizing a complementary strand. Nucleotides are added sequentially, and each incorporation event is detected in real-time, allowing the sequence of the template to be determined. The method is particularly useful for high-throughput sequencing applications where accuracy and speed are critical. The invention improves upon existing sequencing techniques by enabling single-nucleotide resolution, reducing errors, and increasing sequencing efficiency. The system may include components for controlling nucleotide addition, detecting incorporation events, and analyzing the resulting data to reconstruct the nucleic acid sequence. This approach is applicable in genomics, medical diagnostics, and biological research, where accurate and rapid sequencing is essential.
3. The method of claim 1 wherein oligomers are added.
This invention relates to a method for producing a polymer composition with improved properties by incorporating oligomers during the polymerization process. The method addresses the challenge of enhancing polymer performance, such as mechanical strength, thermal stability, or processability, by introducing oligomers that modify the polymer structure at a molecular level. The oligomers are added during polymerization to interact with the growing polymer chains, altering their arrangement and properties. The oligomers may be selected based on their compatibility with the polymer matrix and their ability to enhance specific characteristics, such as flexibility, durability, or resistance to environmental factors. The method ensures uniform distribution of the oligomers within the polymer, preventing phase separation and maintaining material homogeneity. This approach is particularly useful in industries requiring high-performance polymers, such as automotive, aerospace, and packaging, where material properties are critical. The addition of oligomers can also reduce production costs by minimizing the need for post-processing treatments or additives. The resulting polymer composition exhibits superior performance compared to conventional polymers, making it suitable for demanding applications.
4. The method of claim 1 wherein the step of deblocking the 5′ end of the DNA is carried out using a phosphatase enzyme.
This invention relates to a method for processing DNA, specifically focusing on deblocking the 5′ end of DNA molecules. The process involves treating the DNA with a phosphatase enzyme to remove blocking groups or modifications that prevent further enzymatic reactions or sequencing. The method is particularly useful in applications where the 5′ end of DNA must be accessible for subsequent steps, such as ligation, amplification, or sequencing. The use of a phosphatase enzyme ensures efficient and specific removal of phosphate groups or other blocking moieties, improving the yield and accuracy of downstream processes. This technique is applicable in molecular biology, genetic engineering, and DNA sequencing workflows where precise manipulation of DNA ends is required. The method may be combined with other DNA processing steps, such as end repair or adapter ligation, to prepare DNA for high-throughput sequencing or cloning. The invention addresses the challenge of ensuring clean, accessible DNA ends, which is critical for reliable and efficient genetic analysis.
5. The method of claim 1 wherein the DNA is double stranded and further comprising the step of the reserve chamber further comprises a ligase and ATP, to repair the DNA strand not joined by the topoisomerase.
This invention relates to a method for processing double-stranded DNA using a topoisomerase and a ligase to repair and join DNA strands. The method addresses the challenge of efficiently ligating and repairing DNA strands during molecular cloning or genetic engineering processes, particularly when using topoisomerase enzymes that may leave nicks or gaps in the DNA backbone. The process involves a reaction chamber containing a topoisomerase enzyme, which catalyzes the joining of a single DNA strand to a target DNA molecule. The target DNA may have been previously cleaved or prepared for ligation. The reaction chamber also includes a reverse chamber containing a ligase enzyme and ATP, which repairs the complementary DNA strand that was not joined by the topoisomerase. The ligase enzyme catalyzes the formation of a phosphodiester bond, sealing the remaining nicks in the double-stranded DNA. This dual-enzyme approach ensures complete and accurate DNA repair, improving the efficiency and fidelity of DNA assembly processes. The method is particularly useful in applications requiring precise DNA manipulation, such as gene synthesis, CRISPR-based genome editing, or recombinant DNA construction. By combining topoisomerase-mediated strand joining with ligase-mediated repair, the invention provides a robust solution for seamless DNA ligation and repair in molecular biology workflows.
6. The method of claim 1 wherein the topoisomerase-charged donor oligonucleotide comprises a 5′ overhang on the strand complementary to the strand bearing the topoisomerase, comprising a polyinosine sequence.
7. The method of claim 1 wherein the topoisomerase is selected from vaccinia topoisomerase and SVF topoisomerase I.
8. The method of claim 1 wherein a vaccinia topoisomerase which recognizes (C/T)CCTT is used to add deoxythymidine triphosphate (dTTP) nucleotides and a SVF topoisomerase I which recognizes CCCTG is used to add deoxyguanosine triphosphate (dGTP) nucleotides.
9. The method of claim 1 wherein a nanopore separates a chamber comprising the topoisomerase from a chamber comprising a phosphatase, wherein the nanopore is large enough to allow movement of the DNA through the nanopore by electrical attraction but is not large enough to permit movement of the topoisomerase and the phosphatase through the nanopore.
10. The method of claim 1 wherein the DNA is on a substrate or magnetic bead, such that it can be selectively exposed to or removed from the reagents as required to provide the desired sequence.
11. A method of synthesizing a DNA molecule using topoisomerase-mediated ligation, by adding oligomers to a DNA strand in the 3′ to 5′ direction, comprising (i) reacting a DNA molecule with a topoisomerase charged with the desired oligomer, then (ii) removing all of the oligomer thus added with the exception of a single base, using a type IIS restriction enzyme; (iii) dephosphorylating the 5′end of the DNA thus formed using a phosphatase; and (iv) repeating steps (i), (ii) and (iii) until the desired nucleotide sequence is obtained.
12. The method of claim 11 wherein the topoisomerase is selected from vaccinia topoisomerase and SVF topoisomerase I.
13. The method of claim 11 wherein a vaccinia topoisomerase which recognizes (C/T)CCTT is used to add deoxythymidine triphosphate (dTTP) nucleotides and a SVF topoisomerase I which recognizes CCCTG is used to add deoxyguanosine triphosphate (dGTP) nucleotides.
14. The method of claim 11 wherein a nanopore separates a chamber comprising the topoisomerase from a chamber comprising a phosphatase, wherein the DNA can pass through the nanopore by electrical attraction but the topoisomerase and the phosphatase through the nanopore cannot.
15. The method of claim 11 wherein the DNA is on a substrate or magnetic bead, such that it can be selectively exposed to or removed from the reagents as required to provide the desired sequence.
16. A method for synthesizing DNA in a nanochip comprising one or more addition chambers containing an oligonucleotide bound at the 3′ end to a topoisomerase, and one or more reserve chambers containing a phosphatase and optionally a restriction enzyme, said chambers also containing compatible buffer solution and being separated by a membrane comprising at least one nanopore, wherein the enzymes are prevented from passing through the nanopore, comprising (i) moving the 5′ end of a receiver DNA into a first addition chamber, by means of an electrical force, wherein the first addition chamber contains a topoisomerase-charged donor oligonucleotide; (ii) allowing sufficient time for the donor oligonucleotide to ligate to and thereby extend the receiver DNA; (iii) moving the 5′ end of the receiver DNA thus extended into the reserve chamber, by means of an electrical force, wherein the 5′ end of the receiver DNA is dephosphorylated by the phosphatase; and (iv) repeating the cycle of steps (i) (iii), adding oligonucleotides having the same or different informational sequence, until the desired DNA sequence is obtained.
17. The method of claim 16 wherein the enzymes are tethered to a substrate.
18. The method of claim 16 wherein the restriction enzyme is present in the reserve chamber and step (iii) further comprises cleaving of the oligonucleotide added to the receiver DNA by the restriction enzyme at a position which is one nucleotide in the 5′ direction from the oligonucleotide added by topoisomerase, so that each cycle of steps (i)-(iii) adds a single base to the 5′ end of the receiver DNA.
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April 16, 2021
November 22, 2022
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